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Contents:In the table below, Illumina Nextera Handle sequences have been added and highlighted in bold. Primers for the on-target have been added for convenience. The table below is sorted by the CFD off-target score. Sites with very low CFD scores < 0.02 are unlikely to be cleaved, see our study Haeussler et al. 2016, Figure 2.
In the protocol by Matthew Canver, Harvard, two PCRs are run: one PCR to amplify the potential off-target, then a second PCR to extend the handles with Illumina barcodes. Please click here to download the protocol. Alternatively, you can have a look at Fu et al, 2014.
If a primer was not found, the reason is usually that the region around the off-target is too repetitive. To avoid unspecific primers, all repeats are masked for the primer design (not for off-target search). If you think that we should change the parameters here or should use different primer3 settings, please let us know.
| Name | Primer Sequence | Tm | CFD Score |
|---|---|---|---|
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314173_F | TCGTCGGCAGCGTCGAGCCACGGTCTGCTGGAG | 62.6 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314173_R | GTCTCGTGGGCTCGGTGCAGCTCACCTCGATTTCT | 59.3 | 1.00 |
| mm4_intergenic_Gm5973|Gm9915_chr1_41236374_F | Primer3: not found at this Tm | N.d. | 0.57 |
| mm4_intergenic_Gm5973|Gm9915_chr1_41236374_R | Primer3: not found at this Tm | N.d. | 0.57 |
| mm4_intergenic_B230118H07Rik/Rag1|Traf6_chr2_101665039_F | Primer3: not found at this Tm | N.d. | 0.49 |
| mm4_intergenic_B230118H07Rik/Rag1|Traf6_chr2_101665039_R | Primer3: not found at this Tm | N.d. | 0.49 |
| mm4_intergenic_Gm11468|Gm14268_chr2_166323802_F | Primer3: not found at this Tm | N.d. | 0.35 |
| mm4_intergenic_Gm11468|Gm14268_chr2_166323802_R | Primer3: not found at this Tm | N.d. | 0.35 |
| mm4_intergenic_Gm22277|Lrrc30_chr17_67610680_F | Primer3: not found at this Tm | N.d. | 0.30 |
| mm4_intergenic_Gm22277|Lrrc30_chr17_67610680_R | Primer3: not found at this Tm | N.d. | 0.30 |
| mm4_intergenic_Gm9726/mmu-mir-8099-2|Gm26055_chr12_95043868_F | Primer3: not found at this Tm | N.d. | 0.28 |
| mm4_intergenic_Gm9726/mmu-mir-8099-2|Gm26055_chr12_95043868_R | Primer3: not found at this Tm | N.d. | 0.28 |
| mm4_intergenic_Gm5506|Gm5839_chr18_48320896_F | Primer3: not found at this Tm | N.d. | 0.27 |
| mm4_intergenic_Gm5506|Gm5839_chr18_48320896_R | Primer3: not found at this Tm | N.d. | 0.27 |
| mm4_intergenic_Gp9|Rab43_chr6_87780088_F | TCGTCGGCAGCGTCGGCATCTTCATGGCGCTTTT | 59.8 | 0.23 |
| mm4_intergenic_Gp9|Rab43_chr6_87780088_R | GTCTCGTGGGCTCGGGATCAGGGGAGAAGCAGGGA | 60.6 | 0.23 |
| mm3_intergenic_Cntnap5b|Gm25537_chr1_100666275_F | Primer3: not found at this Tm | N.d. | 0.23 |
| mm3_intergenic_Cntnap5b|Gm25537_chr1_100666275_R | Primer3: not found at this Tm | N.d. | 0.23 |
| mm4_intron_Socs5_chr17_87115213_F | TCGTCGGCAGCGTCCTTGGCCACCCGTAAAATGC | 60.1 | 0.21 |
| mm4_intron_Socs5_chr17_87115213_R | GTCTCGTGGGCTCGGCTACTCCGGTGACCCAGAGA | 60.0 | 0.21 |
| mm3_intergenic_Efnb1|Gm14812_chrX_99152905_F | Primer3: not found at this Tm | N.d. | 0.17 |
| mm3_intergenic_Efnb1|Gm14812_chrX_99152905_R | Primer3: not found at this Tm | N.d. | 0.17 |
| mm3_intron_Dcc_chr18_71912709_F | TCGTCGGCAGCGTCCCCAGAAATTCTGCCCAGGT | 59.9 | 0.17 |
| mm3_intron_Dcc_chr18_71912709_R | GTCTCGTGGGCTCGGAGGAAGGGAAGGAAAGGGGA | 59.8 | 0.17 |
| mm4_intergenic_Smpd3|Gm10629_chr8_106279674_F | Primer3: not found at this Tm | N.d. | 0.16 |
| mm4_intergenic_Smpd3|Gm10629_chr8_106279674_R | Primer3: not found at this Tm | N.d. | 0.16 |
| mm4_intron_Slc35b2_chr17_45564854_F | Primer3: not found at this Tm | N.d. | 0.16 |
| mm4_intron_Slc35b2_chr17_45564854_R | Primer3: not found at this Tm | N.d. | 0.16 |
| mm4_intergenic_Igfbp7|Pea15b_chr5_77509528_F | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intergenic_Igfbp7|Pea15b_chr5_77509528_R | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intergenic_Pabpc1|Ywhaz_chr15_36693402_F | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intergenic_Pabpc1|Ywhaz_chr15_36693402_R | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intergenic_Vstm2l|Tti1_chr2_157973820_F | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_intergenic_Vstm2l|Tti1_chr2_157973820_R | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_intergenic_Gm8526|Edil3_chr13_88645854_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intergenic_Gm8526|Edil3_chr13_88645854_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_1700001C02Rik_chr5_30479957_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intron_1700001C02Rik_chr5_30479957_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_9230114K14Rik|Gm25601_chr5_52230884_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_9230114K14Rik|Gm25601_chr5_52230884_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Gm22734|Dlg2_chr7_92405947_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Gm22734|Dlg2_chr7_92405947_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intron_Ptpdc1_chr13_48616777_F | TCGTCGGCAGCGTCTCTGTGTTCACTTATTTGAGGAGT | 57.8 | 0.08 |
| mm4_intron_Ptpdc1_chr13_48616777_R | GTCTCGTGGGCTCGGCGCACTTCTAGCCTGCCTAT | 59.6 | 0.08 |
| mm4_intergenic_Gm5591|Gm6605_chr7_38561777_F | TCGTCGGCAGCGTCCTGTGAGAGGTCCCTGAGGA | 59.9 | 0.08 |
| mm4_intergenic_Gm5591|Gm6605_chr7_38561777_R | GTCTCGTGGGCTCGGCAAAGCCCAGAGTCTGAATTGG | 59.5 | 0.08 |
| mm4_intergenic_Gm25671|Gm16387_chr7_38995320_F | TCGTCGGCAGCGTCCTGTGAGAGGTCCCTGAGGA | 59.9 | 0.08 |
| mm4_intergenic_Gm25671|Gm16387_chr7_38995320_R | GTCTCGTGGGCTCGGCAAAGCCCAGAGTCTGAATTGG | 59.5 | 0.08 |
| mm4_intergenic_Gm23307|Gm5114_chr7_39350533_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Gm23307|Gm5114_chr7_39350533_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm3_intergenic_Gm25740|Gm23481_chr10_46378582_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm3_intergenic_Gm25740|Gm23481_chr10_46378582_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Cdh19|Dsel_chr1_111677021_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Cdh19|Dsel_chr1_111677021_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Foxp1|Gm20696/Foxp1_chr6_99267872_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Foxp1|Gm20696/Foxp1_chr6_99267872_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm24256|Grik2_chr10_48990723_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Gm24256|Grik2_chr10_48990723_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Fhod3_chr18_24880005_F | TCGTCGGCAGCGTCACCTTCTCTGCAGCTCACAC | 59.9 | 0.04 |
| mm4_intron_Fhod3_chr18_24880005_R | GTCTCGTGGGCTCGGCTACCTAGGTGTGGGGCTCT | 60.0 | 0.04 |
| mm4_intergenic_Gm26861|Gm25496_chr13_11121163_F | TCGTCGGCAGCGTCCCGGGCCTCCTTTAATGTGT | 60.0 | 0.04 |
| mm4_intergenic_Gm26861|Gm25496_chr13_11121163_R | GTCTCGTGGGCTCGGGGGCACACTCAGTTGCTCTT | 60.5 | 0.04 |
| mm4_intergenic_Pcdh18|Slc7a11_chr3_49868981_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Pcdh18|Slc7a11_chr3_49868981_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Capn11_chr17_45636325_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Capn11_chr17_45636325_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Aagab_chr9_63603271_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Aagab_chr9_63603271_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Fgf13|Gm14887_chrX_59576677_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Fgf13|Gm14887_chrX_59576677_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Llph|Hmga2/1700006J14Rik_chr10_120352370_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Llph|Hmga2/1700006J14Rik_chr10_120352370_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Coro2b_chr9_62485193_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Coro2b_chr9_62485193_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Aadat_chr8_60529728_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Aadat_chr8_60529728_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Gm26685|Dpt_chr1_164706543_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Gm26685|Dpt_chr1_164706543_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Mdfic_chr6_15744004_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Mdfic_chr6_15744004_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Ddhd2_chr8_25745973_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Ddhd2_chr8_25745973_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Prdm10_chr9_31334188_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Prdm10_chr9_31334188_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Gm25485|Gm12460_chr4_51139130_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Gm25485|Gm12460_chr4_51139130_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Npffr2|Adamts3_chr5_89666452_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Npffr2|Adamts3_chr5_89666452_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Erc2_chr14_27873363_F | TCGTCGGCAGCGTCGGAGTGACATGGAAGGCACA | 59.9 | 0.01 |
| mm4_intron_Erc2_chr14_27873363_R | GTCTCGTGGGCTCGGGTCAGCATACCTCTGACATGGT | 59.8 | 0.01 |
| mm4_intergenic_Gm25382|Cited2_chr10_17534587_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm25382|Cited2_chr10_17534587_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_G930045G22Rik|Mir148a_chr6_50909430_F | TCGTCGGCAGCGTCTGGACAAGAAACCAGGCAGG | 60.1 | 0.01 |
| mm4_intergenic_G930045G22Rik|Mir148a_chr6_50909430_R | GTCTCGTGGGCTCGGCAGGCCTTGTTTGTCAGGGA | 60.1 | 0.01 |
| mm4_intron_Arl15_chr13_113979835_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Arl15_chr13_113979835_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm3_intergenic_Ndufc2|Thrsp_chr7_97409662_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm3_intergenic_Ndufc2|Thrsp_chr7_97409662_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_B130011K05Rik|Gm12287_chr11_63537178_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_B130011K05Rik|Gm12287_chr11_63537178_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Dgat1_chr15_76502228_F | TCGTCGGCAGCGTCAGAACTCCAGGCCCAGGTTA | 60.1 | 0.00 |
| mm4_exon_Dgat1_chr15_76502228_R | GTCTCGTGGGCTCGGCGGTGGCTGTGCTCATGTAT | 60.4 | 0.00 |
| mm4_intergenic_Gm26089|Fat1_chr8_44885802_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm26089|Fat1_chr8_44885802_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Pxk_chr14_8138448_F | TCGTCGGCAGCGTCGTAAGCTGGGCGAGAGGATG | 60.2 | 0.00 |
| mm4_intron_Pxk_chr14_8138448_R | GTCTCGTGGGCTCGGTGCATGTCTCTCTAAGCCAGC | 60.1 | 0.00 |
| mm4_intron_L3hypdh_chr12_72074233_F | TCGTCGGCAGCGTCGGAAAAGGGAGCACATGGGA | 59.9 | 0.00 |
| mm4_intron_L3hypdh_chr12_72074233_R | GTCTCGTGGGCTCGGACTGCCTTATGACTCCAGGT | 58.0 | 0.00 |
| mm4_intergenic_Gm7367|A230006K03Rik_chr7_60939522_F | TCGTCGGCAGCGTCACACAACCACGTGGGAGATG | 60.2 | 0.00 |
| mm4_intergenic_Gm7367|A230006K03Rik_chr7_60939522_R | GTCTCGTGGGCTCGGCAGGAGACCTGTTGCTCCTT | 59.3 | 0.00 |
| mm4_intergenic_Il6ra|Atp8b2_chr3_89935621_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Il6ra|Atp8b2_chr3_89935621_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Aldh1a7|Tmc1_chr19_20732759_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Aldh1a7|Tmc1_chr19_20732759_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Fam129a|Edem3_chr1_151731729_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Fam129a|Edem3_chr1_151731729_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm12722|Gm12723_chr4_105472802_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm12722|Gm12723_chr4_105472802_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_L3mbtl3|AC091763.1_chr10_26353185_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_L3mbtl3|AC091763.1_chr10_26353185_R | Primer3: not found at this Tm | N.d. | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314173 |
GAGCCACGGTCTGCTGGAGCGGCCATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCA ACCTAGTAGCTCAGACACCGAATCGGATTTCTATGAAGAAATCGAGGTGAGCTGCA |
| mm4_intergenic_Gp9|Rab43_chr6_87780088 |
GGCATCTTCATGGCGCTTTTCCCCAATTGGTGTCTGAGCTTCTTTGCAGCATGGGAGGCATGGAGTGGGGGGGGGGGGCT CCCTGCTTCTCCCCTGATC |
| mm4_intron_Socs5_chr17_87115213 |
CTTGGCCACCCGTAAAATGCTGCTTTGGTTGACTGTGAAATTGTGAACCTTAATGATTGTTACCAAGGAAAATGATAGTT AGTAGGGAAGGCACCGAATAGGAGGACTGTCTCTGGGTCACCGGAGTAG |
| mm3_intron_Dcc_chr18_71912709 |
CCCAGAAATTCTGCCCAGGTATAGTAGCTCACACACAGGATGGGGAGTCGGGCACTTTTCATAGTAGTTGGCGAAAGGAT TGTTTCTCCCCTTTCCTTCCCTTCCT |
| mm4_intron_Ptpdc1_chr13_48616777 |
TCTGTGTTCACTTATTTGAGGAGTTGATTTGTCTGTTAAGAACTCTATACTGATCTAAGTAATAGTAACTCAGAAAACAA ATGGGAGGTATAGGCAGGCTAGAAGTGCG |
| mm4_intergenic_Gm5591|Gm6605_chr7_38561777 |
CTGTGAGAGGTCCCTGAGGAAGACACTTAGATTTGATCCTGGCATCTATTCCTAGCTAAGACACTGAATTGGCCAATTCA GACTCTGGGCTTTG |
| mm4_intergenic_Gm25671|Gm16387_chr7_38995320 |
CTGTGAGAGGTCCCTGAGGAAGACACTTAGATTTGATCCTGGCATCTATTCCTAGCTAAGACACTGAATTGGCCAATTCA GACTCTGGGCTTTG |
| mm4_intron_Fhod3_chr18_24880005 |
ACCTTCTCTGCAGCTCACACTCCTGGACTGGAATTGTGTGCCCTGTCCATCCTCCCAGTTCTGTGTCTGAGATACCACCA ACAAGATGGTTTACAGGGAGCAGAGCCCCACACCTAGGTAG |
| mm4_intergenic_Gm26861|Gm25496_chr13_11121163 |
CCGGGCCTCCTTTAATGTGTCTTCAGGACTATAAAGAAGCACAAAAGGAGCTCAGACACTTAATTGGTTTACAGATATGG CAATATGGTAACACTGCAAAAGAGCAACTGAGTGTGCCC |
| mm4_intron_Erc2_chr14_27873363 | GGAGTGACATGGAAGGCACAATCCAGTCTGGAGTCTGAGCTACTATTTACCATGTCAGAGGTATGCTGAC |
| mm4_intergenic_G930045G22Rik|Mir148a_chr6_50909430 |
TGGACAAGAAACCAGGCAGGCTCAGAGCACCTATTAAGGGCAAGCTGGCTTTATGGCATAGCCACCTCGGTTTCTGAGCT CCTATCCTTTATTTTATCCCTGACAAACAAGGCCTG |
| mm4_exon_Dgat1_chr15_76502228 |
AGAACTCCAGGCCCAGGTTAGGGAGGGCTGGCCTTTGGCAGTAGCTCAGACCCCCACTGGGGCATCGTAGTTGAGCACGT AGTAGTCGTGGACATACATGAGCACAGCCACCG |
| mm4_intron_Pxk_chr14_8138448 |
GTAAGCTGGGCGAGAGGATGACAAAAGGAGCCACTTTGCTGTCTGAGCTACTTTCCACAGGCTAATGAGCTGGCTTAGAG AGACATGCA |
| mm4_intron_L3hypdh_chr12_72074233 |
GGAAAAGGGAGCACATGGGAGAGGGTCTAGAAGCTCAGAAACCTAAGAGGGAACTTTAGGCTACACAGCAACATTATGTC TCAAAAAACAAAGAAACTTCTGATTCCTAACTGATACCTGGAGTCATAAGGCAGT |
| mm4_intergenic_Gm7367|A230006K03Rik_chr7_60939522 |
ACACAACCACGTGGGAGATGCTGTCCTGATATAAACACCAATTCCATATCTGATCTACTAAAGCAAATTAAGGAGCAACA GGTCTCCTG |